Rapid and Specific Immunomagnetic Isolation of Mouse Primary Oligodendrocytes

Rafael E. Flores-Obando; Mona M. Freidin; Charles K. Abrams

doi:10.3791/57543

JoVE Journal > Neuroscience

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Neuroscience

Rapido e specifico immunomagnetica isolamento degli oligodendrociti primario del Mouse

Published: May 21, 2018

doi:

10.3791/57543

Rafael E. Flores-Obando¹, Mona M. Freidin², Charles K. Abrams²

¹Program in Molecular and Cellular Biology,State University of New York Downstate Medical Center, ²Department of Neurology and Rehabilitation,University of Illinois at Chicago

Summary

Descriviamo l’isolamento immunomagnetica degli oligodendrociti primario del mouse, che permette l’isolamento rapido e specifico delle cellule per coltura in vitro .

Abstract

L’efficiente e robusto isolamento e coltura dei oligodendrocytes primario (OLs) è uno strumento prezioso per lo Studio in vitro di sviluppo di oligodendroglia, nonché la biologia di demyelinating malattie come la sclerosi multipla e Malattia di Pelizaeus-Merzbacher-simile (PMLD). Qui, presentiamo un semplice e metodo di selezione efficace per l’isolamento di immunomagnetica di fase tre O4⁺ cellule preoligodendrocytes da cuccioli di topi neonatali. Poiché immaturo OL costituiscono più dell’80% della materia bianca roditore-cervello postnatale giorno 7 (P7) questo metodo di isolamento non solo assicura un alto rendimento cellulare, ma anche l’isolamento specifico di OLs già commesso alla stirpe oligodendroglial, diminuendo il possibilità di isolare cellule contaminanti quali gli astrociti e altre cellule del cervello del mouse. Questo metodo è una modifica delle tecniche segnalato precedentemente e fornisce del oligodendrocyte preparazione purezza superiore al 80% in circa 4 h.

Introduction

Oligodendrociti (OLs) sono le cellule di mielinizzazione del sistema nervoso centrale (SNC)¹. L’isolamento e la coltura degli oligodendrociti primari in un ambiente strettamente regolamentato è uno strumento prezioso per lo Studio in vitro di sviluppo di oligodendroglia, nonché la biologia di demyelinating malattie come la sclerosi multipla². Ciò richiede un’efficiente e robusto del oligodendrocyte isolamento e coltura metodo³. In questo studio, abbiamo approfittato dell’espressione di un indicatore della superficie delle cellule del oligodendrocyte distintivo per implementare una tecnica di isolamento modificate che è rapido e specifico.

Sono state identificate quattro distinte fasi di maturazione del oligodendrocyte, ognuna caratterizzata dall’espressione di marcatori di superficie cellulare distintivo per ogni stadio di sviluppo (Figura 1). Questi indicatori di superficie delle cellule possono essere riconosciuti da anticorpi specifici⁴^,⁵e possono essere utilizzati per isolare OLs alle fasi specifiche. Nella prima fase, le cellule del precursore del oligodendrocyte (OPCs) hanno la capacità di proliferare, migrare e rapidi in particolare fattore di crescita derivato dalle piastrine (PDGF-Rα) recettore⁶, ganglioside A2B5, proteoglicano NG2⁷^,⁸, neuro-acido polisialico delle cellule adesione molecola⁹ e grassi-acido-legante proteina 7 (FABP7)¹⁰. Gli OPC hanno morfologia bipolare con alcuni processi brevi che emana dai poli opposti del corpo cellulare, che è caratteristico di cellule precursori neurali¹¹.

Figura 1: espressione di marcatori di superficie delle cellule durante lo sviluppo del oligodendrocyte mouse. OLs cella marcatori di superficie come A2B5, GalC (O1), NG2, O4 e PDGF-Rα può essere utilizzato per isolare specificamente oligodendrociti in fase inerente allo sviluppo specifica utilizzando anticorpi specifici. Clicca qui per visualizzare una versione più grande di questa figura.

Nella seconda fase, OPCs danno luogo a preoligodendrocytes e rapidi alla membrana delle cellule non solo gli indicatori OPC, ma anche il sulfatide (un galattolipide solfonata) riconosciuto dal O4 anticorpo¹²^,¹³e la proteina GPR17¹⁴, che persiste fino alla fase di oligodendrociti immaturi (OL). In questa fase, preoligodendrocytes estendere multipolari processi brevi. Preoligodendrocytes sono la grande fase OL al giorno postnatale 2 (P2) nella materia bianca cerebrale di ratto e topo con una popolazione secondaria di immaturi OLs¹⁵.

Durante la terza tappa, immaturi OLs continuano a esprimere O4, perdere espressione degli indicatori A2B5 e NG2 e cominciano ad esprimere Galattocerebrosidi C¹⁶. In questa fase, OLs sono impegnati alla stirpe oligodendroglial e diventare cellule post-mitotiche con rami lunghi ramificate¹⁷^,¹⁸. Immaturo OL costituiscono più dell’80% della materia bianca del roditore in P7 e in questo momento si osservano le prime cellule MBP⁺ ¹⁵^,¹⁹^,²⁰^,²¹. Di conseguenza, isolamento di OLs presso P7 potrebbe garantire alto rendimento cellulare.

Nella fase finale e la quarta di sviluppo OL, OLs matura esprimono proteine myelinating (proteina basica della mielina (MBP), proteolipide (PLP), glicoproteina mielina associata (MAG) e myelin oligodendrocyte glycoprotein (MOG)²²^,²³ ^,²⁴^,²⁵^,²⁶. In questa fase, OLs matura estendere membrane quello compatto di forma economale guaine intorno gli assoni e sono in grado di mielinare. Questo coincide con l’osservazione che nel cervello di ratto e topo, cellule MBP⁺ diventano sempre più abbondanti a P14¹⁹^,²⁰^,²¹.

Poiché il primo isolamento del oligodendrocyte Fewster e colleghi nel 1967²⁷, sono stati implementati diversi metodi per l’isolamento di OLs da roditore SNC compreso immunopanning²⁸^,²⁹^,³⁰, fluorescenza-attivato delle cellule ordinano (FACS) sfruttando delle cellule degli antigeni di superficie-specific²⁸^,³¹, differenziale mediante centrifugazione in gradiente³²^,³³^,³⁴^,³⁵e agitazione metodo basato sull’adesione differenziale di diverse CNS glia³⁶^,³⁷. Tuttavia, i metodi di coltura esistenti hanno limitazioni, soprattutto in termini di purezza, rendimento e tempo necessario per eseguire la procedure³⁸. Pertanto, i metodi più efficienti di isolamento per gli oligodendrociti sono necessari.

In questa carta, presentiamo un semplice e metodo di selezione efficace per l’isolamento di immunomagnetica di fase tre O4⁺ cellule preoligodendrocytes da cuccioli di topi neonatali. Questo metodo è una modificazione delle tecniche riportate da Emery et al. ³⁹ e Dincman et al. ⁴⁰ e fornisce una purezza di preparazione del oligodendrocyte superiore all’80% in circa 4 h.

Protocol

I topi utilizzati in questo studio sono stati curati per secondo le indicazioni del numero di protocollo di SUNY Downstate Medical Center divisione del laboratorio animale risorse (DLAR) 15-10492. Nota: Oligodendrocytes primario sono stati isolati da neonatale (P5-P7 selvaggio-tipo C57Bl/6N) topi. In questa fase, immaturi OLs costituiscono più dell’80% della materia bianca del roditore garantire alto rendimento cellulare. Tutti i buffer e composizioni di reagente sono disponibili alla fine de…

Representative Results

Lo scopo di questo studio era di stabilire un metodo di isolamento migliorato per O4+ oligodendrociti primario del mouse che richiedono la manipolazione meno possibile delle cellule bersaglio. L’intera procedura dall’eutanasia dei cuccioli di placcatura delle celle di vetrini coprioggetti prende circa 4 h e i dati qui indicati rappresentano tre esperimenti indipendenti. Dopo dissociazione del tessuto, una media di 4,3 ± 0,46 x 107 cellule sono state isolate per ogni…

Discussion

In questa comunicazione, presentiamo un metodo per l’isolamento efficiente delle culture del oligodendrocyte altamente purificata del mouse immaturo. Rispetto ai protocolli precedentemente pubblicati³⁹^,⁴⁰, questo metodo ha reso una purezza più elevata con un livello molto più basso degli astrociti GFAP-positive e una percentuale molto bassa di altre cellule non caratterizzato. È importante sottolineare che questi sono immaturi OLs già commit alla stirpe oligo…

Disclosures

The authors have nothing to disclose.

Acknowledgements

Questo studio è stato sostenuto da sovvenzioni dal National Multiple Sclerosis Society (RG4591A1/2) e il National Institutes of Health (R03NS06740402). Gli autori ringraziano il Dr. Ivan Hernandez e membri del suo laboratorio per fornire consulenza, attrezzature e lo spazio laboratorio.

Materials

10ml serological pipets	Fisher Scientific	13-676-10J
10ml syringe Luer-Loc tip	BD, Becton Dickinson	309604
15ml conical tubes	Falcon	352097
24-well tissue culture plates	Falcon	353935
40µm cell strainer	Fisher Scientific	22368547
50ml conical tubes	Falcon	352098
5ml serological pipets	Fisher Scientific	13-676-10H
60mm tissue culture plates	Falcon	353002
70µm cell strainer	Fisher Scientific	22363548
Alexa Fluor 488 goat anti-mouse IgG (H+L) secondary antibody	Invitrogen	A11001
Alexa Fluor 488 goat anti-rabbit IgM (H+L) secondary antibody	Invitrogen	A21042
Alexa Fluor 488 goat anti-rabbit IgM (H+L) secondary antibody	Invitrogen	A11008
Alexa Fluor 594 goat anti-chicken IgG (H+L) secondary antibody	Invitrogen	A11042
Anti-O4 beads- Anti-O4MicroBeads	Miltenyi Biotec	130-094-543
Apo-Transferrin human	Sigma	T1147
Autofil complete bottle top filter assembly, 0.22um filter, 250ml	USA Scientific	6032-1101
Autofil complete bottle top filter assembly, 0.22um filter, 250ml	USA Scientific	6032-1102
B27 Supplement	Invitrogen	17504-044
Boric acid	Sigma	B7660
Bovine Growth Serum (BGS)	GE Healthcare Life Sciences	SH30541.03
BSA	Fisher Scientific	BP-1600-100
CNTF	Peprotech	450-50
d-Biotin	Sigma	B4639
Desoxyribonuclease I (DNAse I)	Worthington	LS002007
EDTA	Fisher Scientific	S311
Epifluorescence microscope with an Olympus DP70 camera	Olympus	Bx51
Feather disposable scalpels	Andwin Scientific	EF7281C
Forskolin	Sigma	F6886
German glass coverslips, #1 thickness, 12mm diameter round	NeuVitro	GG-12-oz
GFAP antibody	Aves	GFAP
Glucose	Fisher Scientific	D16-1
GlutaMAX	Invitrogen	35050-61
Insulin	Invitrogen	12585-014
Magnetic separator stand – MACS multistand	Miltenyi Biotec	130-042-303
Magnetic separator-MiniMACS separator	Miltenyi Biotec	130-042-302
Millex PES 0.22µm filter unit	Millipore	SLG033RS
Mounting media- Prolong Gold with DAPI	Thermo Fisher	P36930
N-acetyl-cysteine (NAC)	Sigma	A8199
Natural mouse laminin	Invitrogen	23017-015
Neurobasal Medium A	Invitrogen	10888-022
Neurotrophin-3 (NT-3)	Peprotech	450-03
NG2 antibody	Millipore	AB5320
Papain	Worthington	LS003126
PBS without Ca2⁺ and Mg2⁺	Sigma	D5652
PDGF	Peprotech	100-13A
Petri dishes	Falcon	351029
Poly-D-Lysine	Sigma	P6407
Primocin	Invivogen	ant-pm-2
Progesterone	Sigma	P8783
Putrescine	Sigma	P5780
Selection column-LS columns	Miltenyi Biotec	130-042-401
Sodium Selenite	Sigma	S5261
Trace elements B	Corning	25-000-CI
Triiodothyronine (T3)	Sigma	T6397
Triton-X	Sigma	T8787
Trypan Blue Solution	Corning	25-900-CI
Tween 20	Sigma	P1379
B27NBMA			487.75 mL Neurobasal Medium A; 10 mL B27 Supplement; 1 mL Primocin; 1.25 mL Glutamax; Filter sterilize and store at 4 °C until use.
B27NBMA + 10% BGS			27 mL B27NBMA; 3 mL Bovine growth serum
CNTF solution stock (10 µg/ml; 1000X)			Order from Peprotech (450-50). Make up at 0.1 to 1 mg/ml according to Manufacturer’s instruction (may vary from lot to lot) in buffer (e.g. DPBS + 0.2% BSA). Store at -80 °C. Working solution (10 µg/ml, 1000X) 1. Make on 0.2% BSA (Fisher scientific BP-1600-100) in DPBS solution and filter sterilize. 2. Dilute master stock aliquot to 10µg/ml in sterile, chilled 0.2% BSA/DPBS. 3. Aliquot (20µl/tube) and snap freeze in liquid nitrogen. 4. Store aliquots at -80 °C.
d-Biotin stock solution (50 µg/ml; 5000X)			Resuspend d-Biotin (Sigma-B4639) in double-distilled H2O at 50 µg/ml (e.g. 2.5 mg in 50 ml of ddH2O). Resuspension might take fair amount of agitation/vortexing, or mild warming briefly at 37°C. If the d-Biotin still will not solubilize, it is fine to make up a less concentrated (e.g. 10µg/ml), and to add a higher volume to the B27NBMA (1/1000), instead of 1/5000). Store at 4°C.
DNase I stock solution			1. Dissolve at 12,500 U Deoxyribonuclease I / ml in HBSS chilled on ice. 2. Filter sterilize on ice 3. Aliquot at 200 µl and freeze overnight at -20°C. 4. Store aliquots at -20 to -30°C.
Dulbecco’s Phosphate Buffered Saline (w/o Ca2+ and Mg2+)			Dissolve pouch in 1 Liter of water to yield 1 liter of medium at 9.6 grams of powder per liter of medium. Store at 2-8 °C.
Forskolin stock solution (4.2 mg/ml; 1000X)			Add 1 ml of sterile DMSO to 50 mg Forskolin in bottle (Sigma-F6886) and pipette until resuspended. Transfer to a 15 ml centrifuge tube and add 11 ml of sterile DMSO to bring to 4.2 mg/ml. Aliquot (e.g. 20 µl) and store at -20°C.
Hank’s balanced salts (HBSS) (Sigma			1. Measure 900 ml of water (temperature 15-20 °C) in a cylinder and stir gently. 2. Add the power and stir until dissolved. 3. Rinse original package with a small amount of water to remove all traces of the powder. 4. Add to the solution in step 2. 5. Add 0.35 gr of sodium bicarbonate (7.5% w/v) for each liter of final volume. 6. Keep stirring until dissolved. 7. Adjust the pH of the buffer while stirring to 0.1-0.3 units below pH= 7.4 since it may rise during filtration. The use of 1N HCl or 1N NaOH is recommended to adjust the pH. 8. Add additional water to bring the final volume to 1L. 9. Sterilize by filtration using a membrane with a porosity of 0.22 microns. 10. Store at 2-8 °C.
Insulin stock solution (4000 µg/ml)			Thaw the bottle and aliquot 25 µl per microcentrifuge tube and store at -20°C.
Laminin solution			Slowly thaw laminin in the cold (2°C to 8°C) to avoid gel formation. Then, aliquot into polypropylene tubes. Store at 5° C to -20° C in aliquots (e.g. 20 µl) and do not freeze/thaw repeatedly. Laminin may be stored at these temperatures for up to six months.
Magnetic Cell Sorting (MCS) Buffer			Prepare the solution containing phosphate-buffered saline (PBS), pH 7.2, and 0.5% bovine serum albumin (BSA), 0.5 mM EDTA, 5µg/ml Insulin, 1 g/L Glucose. Sterilize and degas by filtration the buffer by passing it through a 0.22 µm Millex filter. Store the buffer at 4°C until use
N-Acetyl-L-cysteine (NAC) stock solution (5mg/ml; 1000X)			Dissolve N-Acetyl-L-cysteine (Sigma-A8199) at 5 mg/ml in DMEM (e.g. 50 mg NAC in 10 ml B27NBMA). Filter sterilize and aliquot (e.g. 20 µl). Store at -20°C.
NT3 stock solution (1 µg/ml; 1000X)			Master stock: Order from Peprotech (450-03). Make up at 0.1 to 1 mg/ml according to manufacturer’s instructions (may vary from lot to lot), in buffer (e.g. DPBS + 0.2% BSA). Store at -80°C. Working stock (1µg/ml; 1000X): 1. Make on 0.2% BSA in DPBS solution and filter sterilize. 2. Dilute master stock aliquot to 1 µg/ml in sterile, chilled 0.2% BSA/DPBS. 3. Aliquot (e.g. 20µl/tube) and snap freeze in liquid nitrogen. 4. Store aliquots at -80°C.
PDGF stock solution (10 µg/ml; 1000X)			Master stock: Order from Peprotech (100-13A). Make up at 0.1 to 1 mg/ml according to manufacturer’s instructions (may vary from lot to lot) in buffer (e.g. DPBS) + 0.2% BSA). Store at -80°C. Working stock (1µg/ml; 1000X): 1. Make on 0.2% BSA in DPBS solution and filter sterilize. 2. Dilute master stock aliquot to 1µg/ml in sterile, chilled 0.2% BSA/DPBS. 3. Aliquot (e.g. 20µl/tube) and snap freeze in liquid nitrogen. 4. Store aliquots at -80°C.
Poly-D-lysine (1mg/ml; 100X)			Resuspend poly-D-lysine, molecular weight 70-150 kD (Sigma P6407) at 0.5mg/ml in 0.15M boric acid pH 8.4 (e.g. 50mg in 50ml borate buffer). Filter sterilize and aliquot (e.g. 100µl/tube). Store at -20°C. Prior to use, dilute the 100X stock (1mg/ml) to 50 µg/ml in sterile water.
Oligodendrocyte proliferation media			see Supplementary Table 1
Oligodendrocyte differentiation media			see Supplementary Table 1
Sato supplement (100X)			see Supplementary Table 1
References: the list of reagents and recipes were adopted from the protocols previously described by Emery et. al. 2013 (Emery, B. & Dugas, J. C. Purification of oligodendrocyte lineage cells from mouse cortices by immunopanning. Cold Spring Harb Protoc. 2013 (9), 854-868, doi:10.1101/pdb.prot073973, (2013)) and Dincman et. al. (Dincman, T. A., Beare, J. E., Ohri, S. S. & Whittemore, S. R. Isolation of cortical mouse oligodendrocyte precursor cells. J Neurosci Methods. 209 (1), 219-226, doi:10.1016/j.jneumeth.2012.06.017, (2012))

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Flores-Obando, R. E., Freidin, M. M., Abrams, C. K. Rapid and Specific Immunomagnetic Isolation of Mouse Primary Oligodendrocytes. J. Vis. Exp. (135), e57543, doi:10.3791/57543 (2018).

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